1. Field of the Invention
This invention relates to gene manipulation, and more specifically, relates to a DNA containing a gene participating in the production of L-homoglutamic acid (also referred to as L-2-amino-adipic acid or L-α-aminoadipic acid), and a production system of L-homoglutamic acid (hereinafter, merely referred to as homoglutamic acid) using it.
2. Description of the Related Art
Homoglutamic acid is found widely in organisms such as plants including Cholera vibrio as a bacterium and corn (Zea mays), the embryos of frogs. Homoglutamic acid acts as an intermediate of lysine biosynthesis in fungi, etc. and as a precursor in biosynthesis of β-lactam antibiotics. Further, homoglutamic acid is also useful as a synthetic intermediate of various medicines including methotrexate derivatives (WO 92/09436).
Since preparation of homoglutamic acid by chemical synthesis needs optical resolution and multistage reaction, it is not a useful means from the aspect of costs. On the other hand, a process of preparing homoglutamic acid from L-lysine using a microorganism belonging to the genus Agrobacterium, Klebsiella, Alcaligenes, Brevibacterium or Bacillus is known (Japanese Laid-open Patent Publication No. 6-181787). Part of the present inventors also proposed a process of preparing homoglutamic acid from L-lysine using a microorganism belonging to the genus Flavobacterium (WO 96/31616). However, even in the process using such a microorganism, a process capable of preparing homoglutamic acid more efficiently is desired earnestly.
Thus, the present inventors aimed to reinforce the production system of homoglutamic acid in any of the above microorganisms, for example by gene manipulation. When a review of helpful information is made on the manipulation, for example, as part of researches of biosynthetic pathway of cephamycin C, are confirmed the presence of lysine-6-aminotransferase and L-Δ1-piperidine-carboxylate dehydrogenase participating in conversion from L-lysine to α-aminoadipic acid (or homoglutamic acid) of Streptomyces clavuligerus as a cephamycin C-producing actinomycetes, and as to the former, the presence position of the gene encoding the enzyme, etc. (Fuente et al., Biochem. J. (1997) 327, 59-64).
As to Flavobacterium lutescens (which was re-identified from Flavobacterium fuscum) IFO 3084 used in bioassay of L-lysine, it is known that 2-oxoglutarate 6-aminotransferase [or lysine 6-aminotransferase (hereinafter also referred to as LAT)] catalyzing the following pathway is present (Soda et al., Biochemistry 7 (1968), 4102-4109, ibid. 4110-4119).

In the above bioassay, the absorbance of the product obtained by reacting piperidine-6-carboxylic acid (hereinafter, also referred to as P6C) with o-aminobenzaldehyde is measured. In another bioassay of L-lysine, the L-lysine 6-dehydrogenase activity of Agrobacterium tumefaciens is utilized (Misono et al., J. Biochem. (Tokyo) 105 (1989), 1002-1008).
The above IFO 3084 strain is commonly used in bioassay of L-lysine as mentioned above, and its use method is also established. Therefore, if the IFO 3084 strain had a gene encoding a protein having P6C (or, the 2-aminoadipic acid semialdehyde which is said to be in a quantitatively equilibrium state with P6C in a living body) dehydrogenase (hereinafter, also merely referred to as dehydrogenase) activity, in addition to LAT, the strain would be a candidate bacterium for gene cloning meeting the object of the present invention, namely the object to provide a gene participating in the production of homoglutamic acid.